Development of a new slice-culture method to study epithelial corneal pathophysiology in the paradigm of...

The cornea is a physiologically capillary-free and optically transparent tissue that subserves optimal visual function. In frequent pathological situations of diverse natural or experimental etiologies (lesions, burns, denervations, dystrophies, ulcers, infections) that involve long-lasting epithelial defects, vascular capillaries invade the cornea, resulting in opacity and reduction or loss of visual function. In view of the relative insensitivity of animal models, of the need for large numbers of animals to perform experiments, and, not least, of the painful interventions due to the very dense trigeminal innervation of the cornea in situ, we searched for alternative experimental setups to study mechanisms of corneal pathophysiology. As a first goal we developed a new slice-culture model and examined interactions between in-vitro-differentiated corneal epithelial cells and cocultured vascular endothelial cells. Corneas from enucleated eyes, from donor eyes retrieved for keratoplasty, or from animal eyes were dissected and cut along the optical axis to produce slices containing all three corneal layers. Corneal epithelial cells developed well-differentiated histotypical monolayers whose sizes were comparable with the size of the corneal surface in situ. When examined by electron microscopy, the epithelial cells formed tight contacts with each other and formed typical villous protrusions at the growing front. As a second goal we studied interactions between corneal epithelium and cocultured capillary endothelial cells. Upon encountering cocultured vascular endothelial cells, corneal epithelium exerted a strong, contact-mediated repulsion. The morphological correlates of repulsion were collapse of lamellipodial protrusions, immobilization, and cell death. These responses resulted in the inability to penetrate the corneal epithelium. The repulsive activity was exclusively localized in the epithelium, whereas stromal keratocytes were permissive for ingrowth of vascular cells. The angiorepulsive features of the cornea could be partially neutralized with aprotinin, indicating that membrane-linked proteolytic systems are involved in the interactions between corneal and vascular tissue. This coculture system may be used to characterize further the cell-signaling mechanisms and the interactions between cornea and vascular endothelial cells during pathological situations associated with angiogenesis.